CN105555947A - High expression zymomonas promoters - Google Patents
High expression zymomonas promoters Download PDFInfo
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- CN105555947A CN105555947A CN201480051856.XA CN201480051856A CN105555947A CN 105555947 A CN105555947 A CN 105555947A CN 201480051856 A CN201480051856 A CN 201480051856A CN 105555947 A CN105555947 A CN 105555947A
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Abstract
Synthetic, derivative promoters for expression of chimeric genes in Zymomonas, Zymobacter, and related bacteria were created. The promoters have a C to T change in the nucleotide at position 90 of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter. Promoters with this change have higher expression as compared to the native promoter from which they are derived and are useful for genetic engineering for expressing coding regions or regulatory RNA to obtain high levels of expressed proteins or regulatory RNAs.
Description
Technical field
The present invention relates to microbiology and genetically engineered field.More specifically, identify for guiding the Novel promoter that in bacterium, mosaic gene is expressed.
Background technology
The alcohol production of microorganism provides the alternative energy of fossil oil and therefore becomes the key areas of current research.Zymomonas mobilis (Zymomonasmobilis) is the natural ethanologenic organisms that can be used for business alcohol production.But do not utilize wood sugar under zymomonas mobilis its natural environment, it is pentose main in hydrolysis of lignocellulose material.People wish that ethanologenic organisms utilizes wood sugar to make full use of the carbohydrate substrates in lignocellulose biomass material hydrolyzate.
Other ethanologenic organisms of wood sugar is not utilized to carry out the genetically engineered for wood sugar by introducing gene, described genes encoding 1 under zymomonas mobilis and its natural environment) xylose isomerase, its catalysis wood sugar is to the conversion of xylulose; 2) xylulokinase, it makes xylulose phosphate to form xylulose-5-phosphoric acid; 3) transketolase; With 4) transaldolase.For the through engineering approaches (US5 of xylose metabolism to Zymomonas mobilis strain, 514,583, US5,712,133, US6,566,107, US5, the people such as 726,053, Feldmann (1992) ApplMicrobiolBiotechnol38:354-361, the people such as Zhang (1995) Science267:240-243), and the through engineering approaches (people (2007) Appl.Environ.Mirobiol.73:2592-2599 such as Yanase) of Zymobactef bacillus (Zymobacterpalmae) bacterial strain is had successfully.But, growth on wood sugar of the bacterial strain of through engineering approaches and produce ethanol usually be not as equally good as glucose.
To this through engineering approaches, express the gene of coding for the heterologous protein of xylose metabolism by promotor activated in zymomonas mobilis cell, described promotor is generally the promotor of zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene or the promotor of zymomonas mobilis enolase gene.The bacterial strain carrying out through engineering approaches for xylose utilization is tamed by continuous passage on xylose media, creates the bacterial strain of the xylose utilization with improvement, as described in US7223575 and US7741119.US7989206 and US7998722 discloses the zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter and its that have and improve activity in fermentation single cell bacterium (Zymomonas), expresses the purposes of xylose isomerase for xylose utilization.
Still have demand for other strong promoter, described other strong promoter can be used for the genetically engineered of fermentation single cell bacterium and other ethanologenic organisms to express the gene giving the xylose utilization improved.Described promotor also can be used for the expression of other gene.
Summary of the invention
The invention provides to have and improve active synthetic promoter, it is the derivative of zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter.
Therefore, the invention provides the nucleic acid molecule of synthesis, it comprises the zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter having and replace the base of C at position 90 place T; Wherein said Position Number belongs to SEQIDNO:1.Compared with natural motion fermentation single cell bacterium glyceraldehyde-3-phosphate dehydrogenase gene promoter, the replacement of this base has the effect increasing described promotor intensity.
In other side, the invention provides the mosaic gene of the nucleic acid molecule comprising synthesis and comprise its carrier, the nucleic acid molecule of described synthesis has above-mentioned base and replaces.
In other, the invention provides the method for transforming bacterial cells, described bacterial cell is selected from Zymomonas cells and fermentation bacillus (Zymobacter) cell, and described method comprises and being incorporated in described cell by synthetic nucleic acid molecule of the present invention.
On the other hand, the invention provides the recombinant bacteria bacterial strain comprising synthetic nucleic acid molecule of the present invention, described recombinant bacteria bacterial strain is selected from fermentation single cell bacterium and fermentation bacillus.
In another embodiment, the invention provides the method for the production of the synthetic glycerine aldehyde-3-phosphate dehydrogenase gene promoter improved, the method comprises:
A) be always separated with the glyceraldehyde-3-phosphate dehydrogenase gene of fermentation bacillus the nucleic acid fragment comprising glyceraldehyde-3-phosphate dehydrogenase gene promoter region from fermentation single cell bacterium;
B) at position 90 place T, the base of C will be replaced to the isolated fragment be incorporated into a), thus produce the synthetic glycerine aldehyde-3-phosphate dehydrogenase gene promoter improved.
accompanying drawing illustrates and sequence description
Fig. 1 shows the plasmid map of pZP13.
Subsidiary sequence description according to detailed description below and formation the application part can understand the present invention more fully.
One sequence meets 37C.F.R.1.821-1.825 (" requirement-sequence rules to containing nucleotide sequence and/or aminoacid sequence disclosure ") and meets the sequence listing requirements (rule 5.2 and 49.5 (a-bis), and 208 joint and appendix C of administrative instruction) of World Intellectual Property Organization (WIPO) standard ST.25 (2009) and EPO and PCT.The regulation shown in 37C.F.R. § 1.822 is followed for the symbol of Nucleotide and amino acid sequence data and form.
SEQIDNO:1 is the nucleotide sequence of the natural ZmPgap of zymomonas mobilis CP4 bacterial strain.[in 4236 #1]
SEQIDNO:2 is the nucleotide sequence of the ZmPgap of the zymomonas mobilis CP4 bacterial strain at position 90 place with C to T change.
SEQIDNO:3 is the nucleotide sequence of the natural ZmPgap of the ZM4 bacterial strain of zymomonas mobilis.[in 4236 #2]
SEQIDNO:4 is the nucleotide sequence of the ZmPgap of the zymomonas mobilis ZM4 bacterial strain at position 90 place with C to T change.
SEQIDNO:5 is the nucleotide sequence of the ZmPgap of pZB4.
[in 4236, T lacks #3]
SEQIDNO:6 is the nucleotide sequence of the ZmPgap of the pZB4 at position 90 place with C to T change.
SEQIDNO:7 is the nucleotide sequence of the ZmPgap of zymomonas mobilis ZM4 bacterial strain, and it has T disappearance (as in pZB4) and has the change of C to T at position 90 place at position 285 place.
SEQIDNO:8 is the nucleotide sequence of 1,951-bpDP1341-U fragment.
SEQIDNO:9 is the nucleotide sequence of 1,255-bpDP1341-U fragment.
SEQIDNO:10 is the nucleotide sequence of GUS encoding sequence.
SEQIDNO:11 is the nucleotide sequence of intestinal bacteria (E.coli) araD3 ' UTR.
SEQIDNO:12 is the nucleotide sequence of Spec-R expression cassette.
SEQIDNO:13 is the nucleotide sequence of pZP1332.
SEQIDNO:14 is the nucleotide sequence of pZP1337.
SEQIDNO:15 is the nucleotide sequence of loxPw fragment, and it is positioned at 5 ' of described Spec-R expression cassette.
SEQIDNO:16 is the nucleotide sequence of loxPw fragment, and it is positioned at 3 ' of described Spec-R expression cassette.
The flank loxPw sequence comprising loxPw site and restriction site is SEQIDNO:15 and 16.
Embodiment
Following definition can be used to explain claims and specification sheets:
As used herein, term " comprises ", " comprising ", " comprising ", " comprising ", " having ", " having ", " containing " or " containing ", or their other modification any is intended to contain comprising of nonexcludability.Such as, the composition of containing element list, mixture, technique, method, goods or equipment need not be only limitted to those elements, and other elements clearly do not listed can be comprised, or the element that such composition, mixture, technique, method, goods or equipment are intrinsic.In addition, unless expressly stated on the contrary, "or" refers to the "or" of inclusive and the "or" of nonexcludability.Such as, condition A or B meets following any one: A is real (or existence) and B is false (or non-existent), A is false (or non-existent) and B is real (or existence), and A and B is really (or existence).
In addition, the indefinite article of number before element of the present invention or component " " or " one " that relate to the example (namely occurring) of element or component are intended to for nonrestrictive.Therefore, " one " or " one " should be interpreted as and comprise one (kind) or at least one (kind), and the word singulative of element or component also comprises plural, obviously represents odd number unless there are numeral.
As used herein, term " invention " or " the present invention " are non-limiting terms, and are not intended to mean any independent embodiment of the present invention, but contain all possible embodiment as described in this specification sheets and claim.
As used herein, the term " about " that the amount of modifying composition of the present invention or reactant uses refers to the change of generable umerical amount, such as: in real world, by for the preparation of enriched material or use the general measure of solution and fluid treatment operation; By unintentional error in these operations; By the difference in the manufacture of the composition for the preparation of composition or manner of execution, source or purity; Etc..Term " about " also comprises the different amount due to the different equilibrium conditions relative to the composition by specific starting mixt gained.No matter whether modified by term " about ", the equal parts of the claim amount of comprising.In one embodiment, term " about " refers within report numerical value 10%, preferably within report numerical value 5%.
Term " xylose isomerase " refers to the enzyme of the change of catalysis D-wood sugar and D-xylulose.Xylose isomerase (XI) belongs to the group of the enzyme classifying as EC5.3.1.5.
Term " xylulokinase " refers to that phosphorylation xylulose forms the enzyme of xylulose-5-phosphoric acid.
Term " transketolase " and " transaldolase " refer to two kinds of enzymes in pentose phosphate approach, and xylulose-5-phposphate is become to connect the intermediate of pentose metabolism and Entner-Douderoff glycolytic pathway by it, and this approach makes xylose metabolism become ethanol.
Term " gene " refers to the nucleic acid fragment of expressing specified protein or functional RNA molecule, its optionally comprise be positioned at encoding sequence before (5 ' non-coding sequence) and regulating and controlling sequence afterwards (3 ' non-coding sequence)." natural gene " or " wild type gene " refers to the naturally occurring gene with himself regulating and controlling sequence." mosaic gene " refers to it is not any gene of natural gene, is not the regulating and controlling sequence and encoding sequence existed together under being included in its natural environment.Therefore, mosaic gene can comprise the regulating and controlling sequence and encoding sequence that come from different sources, or comprises and come from same source but to be different from the regulating and controlling sequence and encoding sequence that naturally occurring mode arranges." native gene " refers to the natural gene being in its natural place in biological genome." alien gene " refers to the gene be not present under normal circumstances in host living beings, but it is incorporated in host living beings by transgenosis.Alien gene can comprise the natural gene be inserted in non-native organism, or mosaic gene.
Term " promotor " refers to can control coding sequence or the functional r NA DNA sequence dna of expressing.In general, encoding sequence is positioned at 3 ' end of promoter sequence.Promotor wholely can come from natural gene, or is made up of the different elements coming from naturally occurring different promoters, or even comprises the DNA fragmentation of synthesis.Those skilled in the art should be appreciated that different promotors can in different tissues or cell type, or in the different etap, or respond the expression of different envrionment conditionss and guiding gene.Usually the promotor of genetic expression is caused to be called " constitutive promoter " by most cell types, in most of the cases.
As used herein, term " expression " refers to transcribing and stablely to gather derived from the coding (mRNA) of gene or functional r NA.Express and mRNA also can be referred to translate into polypeptide." process LAN " refers to that the gene product produced in genetically modified organism exceeds the level of the gene product produced in normal bio or unconverted biology.
As used herein, term " conversion " refers to nucleic acid fragment to be transferred in host living beings, causes genetic stability on gene.The nucleic acid shifted can be the plasmid form retained in host cell, or the nucleic acid of some transfer can be integrated in described host cell gene group.It is biological that host living beings containing the nucleic acid fragment shifted to some extent is called as " transgenosis " or " restructuring " or " conversion ".
As used herein, term " plasmid " and " carrier " refer to the extra-chromosomal element of the gene usually carrying the part not belonging to cell centre metabolism, and the form of normally circular double stranded DNA molecule.This class component can be the nucleotide sequence (linear or ring-type) being derived from the autonomously replicating sequence in any source, genome integration sequence, phage or strand or double-stranded DNA or RNA, wherein multiple nucleotide sequence has connected or has recombinated and entered in unique design body, and the promoter fragment of selected gene product can be introduced in cell together with suitable 3 ' non-translated sequence with DNA sequence dna by this unique design body.
Term " is operably connected " association of the nucleotide sequence referred in mononucleotide fragment, makes the function of one of them nucleotide sequence be subject to the impact of another nucleotide sequence.Such as, when promotor can affect expression (that is, what described encoding sequence was subject to promotor transcribes control) of encoding sequence, then this promotor is operably connected with this encoding sequence.Encoding sequence can may be operably coupled to regulating and controlling sequence by the orientation of sense or antisense.
Term " selected marker " refers to a kind of qualification factor, normally microbiotic or pharmaceutical chemicals resistant gene, this factor can based on the effect of marker gene, namely, antibiotic resistance is selected, wherein said effect for follow the trail of heredity concerned nucleic acid and/or for the identification of heredity the cell of concerned nucleic acid or biology.
As used herein, term " Codon degeneracy " refers to the character of the genetic code changed when allowing nucleotides sequence to be listed in the aminoacid sequence not affecting coded polypeptide.Technician will fully recognize: " codon preference " that particular host cell shows when using nucleotide codon to determine given amino acid.Therefore, when synthetic gene is in order to improve the expression in host cell, ites is desirable to design gene, make its codon usage frequency close to the preferred codon usage frequency of this host cell.
Term " codon optimized " is when it relates to gene or the coding region of the nucleic acid molecule for transforming different hosts, referring to when not changing the polypeptide by DNA encoding, changing codon in the gene of nucleic acid molecule or coding region reflect the common codon use of host living beings.
Term " allos " refers to that non-natural is present in concerned position.Such as heterologous gene refers to the gene be still introduced in by transgenosis in host living beings that in host living beings, non-natural exists.Such as, the exogenous nucleic acid molecule be present in mosaic gene is that the non-natural relevant to other mosaic gene fragment exists nucleic acid molecule, such as, have the nucleic acid molecule of coding region that non-natural is each other correlated with and promoter fragment.
As used herein, " nucleic acid molecule of synthesis " is the polymkeric substance of RNA or DNA that are artificial or regulation and control, and it is strand-or double-strand, optionally comprise synthesis, non-natural-or the nucleotide base that changes.The nucleic acid molecule of the synthesis of DNA polymer form can be made up of the fragment of one or more cDNA, genomic dna or synthetic DNA.
Term " zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter " and " ZmPgap " refer to the nucleic acid molecule with promoter activity, and it has the nucleotide sequence of the natural glyceraldehyde-3-phosphate dehydrogenase upstream of coding region be present in described zymomonas mobilis gene.These terms refer to the promotor of Zymomonas mobilis strain, such as CP4 and ZM4 bacterial strain (being respectively SEQIDNO:1 and 2) and refer to the variant in sequence and/or length, it there is no guiding expression in different levels, the ZmPgap (SEQIDNO:3) of such as pZB4 substantially.
Standard recombinant dna used herein and molecule clone technology are known in the art, and have in such as Publication about Document and more fully describe: Sambrook, J., Fritsch, E.F. and Maniatis, T., MolecularCloning:ALaboratoryManual, the 2nd edition; ColdSpringHarborLaboratory:ColdSpringHarbor, NewYork, 1989 (hereinafter referred to as " Maniatis "); And Silhavy, T.J., Bennan, M.L. and Enquist, L.W., ExperimentswithGeneFusions; ColdSpringHarborLaboratory:ColdSpringHarbor, NewYork, 1984; And the people such as Ausubel, F.M, CurrentProtocolsinMolecularBiology, is published by GreenePublishingandWiley-Interscience, and 1987.
The present invention relates to the synthesis expressed for mosaic gene in fermentation single cell bacterium, fermentation bacillus and relevant bacterium and derive promotor.Described promotor with its derive compared with the natural promoter of originating there is higher expression, and can be used for the genetically engineered for expressing coding region or rna regulation carry out to obtain the protein of high level expression or rna regulation in fermentation single cell bacterium, fermentation bacillus and relevant bacterium.Such as, the Zymomonas cells xylose utilization that this promotor can be used for genetically engineered to be improved, it is undertaken, such as xylose isomerase by expressing the coding region of the enzyme of xylose utilization approach.
the glyceraldehyde-3-phosphate dehydrogenase gene promoter improved
This promotor is the derivative of the promotor from described zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene (ZmPgap), it has the replacement of T to C in position 90 place of ZmPgap, and position 90 refers to the Nucleotide+90 in SEQIDNO:1.The promotor with the change of this Nucleotide has the activity of raising compared with natural ZmPgap.
In one embodiment, this promotor has the nucleotide sequence of SEQINNO:2, and except the T at position 90 place, it is identical with SEQIDNO:1.
In many embodiment:, with regard to the Nucleotide of SEQIDNO:1, this promotor has any ZmPgap of T to the replacement of C at position 90 place.The sequence of ZmPgap is not single sequence, but some can be had in sequence not have the variation of materially affect to promoter function.Described promoter sequence has guided the expression level be substantially similar to by being present in the expression level that ZmPgap guides in natural motion Zymomonas strain not have materially affect to mean to promoter function.The variation of sequence can naturally be present between zymomonas mobilis different isolates or bacterial strain, such as with regard to the Nucleotide of SEQIDNO:1 in the place of position 276 described CP4 and ZM4 ZmPgap between difference, this position be A in CP4 and be G (respectively SEQIDNO:1 and 3) in ZM4.In one embodiment, this promotor has the sequence of the ZmPgap of described ZM4 bacterial strain, and it has the replacement (SEQIDNO:4) of T to C at position 90 place.
In addition, in ZmPgap sequence, also can there be one or more insertions or the disappearance that promoter function be there is no to materially affect, such as, be present in pZB4 carrier.With regard to the Nucleotide of SEQIDNO:1, the ZmPgap of described pZB4 carrier, at position 285 place disappearance T, creates SEQIDNO:5.As described in CP4 bacterial strain this promotor at position 276 place, there is A.In one embodiment, this promotor has the sequence of the ZmPgap of described pZB4 carrier, and it has the replacement (SEQIDNO:6) of T to C at position 90 place.
One or more insert or lack the Position Number that can affect relative to SEQIDNO:1, and this depends on whether described insertion or disappearance are present in described promotor 5 ' end and SEQIDNO:1 between the place of position 90.Therefore, the change in location of C to T carries out best identified by contiguous sequence.The contiguous sequence of the change of described C to T is: CAGGGA
cgACAAT, wherein the C of runic and band underscore is the Nucleotide becoming T in this derivative promotor.Those skilled in the art should be easy to be identified in this Nucleotide place change from C to T (also referred to as the replacement of T to C), and this place is called position 90 with regard to SEQIDNO:1.
In another embodiment, this promotor has the replacement of T to C at position 90 place, at position 285 place, there is disappearance T (as in pZB4) and have at position 276 place G (as described in ZM4 bacterial strain) (SEQIDNO:7).Position 90 place have T to the replacement of C, at position 285 place, there is disappearance T (as in pZB4) and have at position 276 place A (as described in CP4 bacterial strain) promotor identical with SEQIDNO:6.All positions are relative SEQIDNO:1.
Therefore, the example of described promotor comprises SEQIDNO:2,4,6 and 7.Comprise and givenly above above at position 90 place, there is the ZmPgap of T to the replacement of C and be referred to herein as PgapU or P
gapU.
the PgapU that preparation improves
By any means known to those skilled in the art, described position 90 place is replaced in introducing ZmPgap nucleic acid molecule.Such as, the oligonucleotide with described sudden change and neighbouring DNA sequence dna can be synthesized and cloned and enter larger promoter dna fragment, to replace not containing the fragment that described position 90 place replaces.Can synthesize comprise described Nucleotide change and some adjacent promoters sequences primer and use it for PCR to prepare described promoter fragment.Whole promoter dna fragment can be synthesized the multiple oligonucleotide linked together.Rite-directed mutagenesis can be used to replace to introduce described Nucleotide.
the Pgap improved in mosaic gene and carrier, to the introducing in bacterial cell
This promotor can be may be operably coupled to the exogenous nucleic acid molecule of expressing in bacterial cell, form chimeric nucleic acid molecule or the mosaic gene of the present invention's another aspect.The design of described mosaic gene and structure are well known to those skilled in the art.Mosaic gene generally includes promotor, and exogenous nucleic acid molecule to be expressed and 3 ' stops control region.Termination control region can come from several genes and usually take from target host cell is natural gene.The described exogenous nucleic acid molecule be operably connected can be any nucleic acid molecule, and it expects to express in bacterial cell, comprises such as, the coding region of protein or peptide or the nucleic acid for function RNA expression.Function RNA comprises such as, sense-rna, ribozyme and RNA interfering.In addition, also can build Operand, it comprises promotor as herein described and the multiple coding regions by described promoter expression.
Promotor as herein described can be used for mosaic gene, and it in order to express in the bacterium belonging to fermentation single cell bacterium or fermentation bacillus.Mosaic gene can be used to carry out the expression of arbitrary protein matter, and described protein participates in the generation of fermentation single cell bacterium or fermentation bacillus product.Such as, the mosaic gene by having these promotors expresses one or more enzymes participating in amino acid (such as L-Ala) or sorbyl alcohol or Xylitol synthesis.Can not utilize in the natural fermented Zymomonas mobilis of wood sugar or fermentation bacillus strain or at wood sugar-utilize in bacterial strain and express described mosaic gene.Promotor as herein described also can be used to carry out the expression of the enzyme relating to xylose metabolism or another pathways metabolism.
Mosaic gene as herein described usually builds in the carrier or transfers in carrier for further operation.Carrier is well known in the art.Some carrier can copy and shift by engaging in host bacteria widely.PRK404 and three kind of related vector: pRK437, pRK442 and pRK442 (H) the complete and sequence of band annotation is available.Prove that these derivatives are the useful tools (people such as Scott, Plasmid50 (1): 74-79 (2003)) in the genetic manipulation carried out in Gram-negative bacteria.
Other carrier known can be used for different target host cells.What be particularly useful for the expression in fermentation single cell bacterium is the carrier of holding concurrently and can copy in intestinal bacteria and fermentation single cell bacterium, such as, at US5, and the pZB188 described in 514,583.Carrier can be included in cell the plasmid stablizing self-replicating and the plasmid carrying the construct in bacterial genomes to be incorporated into.The plasmid integrated for DNA can comprise transposon, support with the nucleotide sequence district of target bacteria genome homology or other sequence integrated.The carrier of other type also swivel base body, such as, its use by
commercially available available system and producing.How suitable carrier is selected to be well-known for required target host and required function.
Also can when do not have be operably connected for express nucleic acid molecule promotor as herein described to be building up in carrier and to be incorporated into contiguous endogenous coding region to replace the endogenesis promoter in bacterial genomes, such as, or interpolation promotor, makes an addition to the coding region in Operand.The replacement of WITH A can use following methods to complete, such as, the method described by people (Can.J.Microbiol. (2007) 53:56-62) such as people (Metab.Eng. (2006) 8:79-90) and White such as Yuan.
The carrier comprising promotor as herein described is incorporated in bacterial cell by the method by knowing, such as, use freeze thawing conversion, calcium mediated transformation, electroporation or joint.
pgapU is used to carry out the expression of exogenous nucleic acid molecule
The mosaic gene that the Pgap of improvement as herein described can be used to obtain improve the standard is expressed.As shown in present example 3, compared with the activity by protein expressed by natural Pgap, the mosaic gene comprising this promotor that may be operably coupled to coding region gives the activity improving protein expressed by more than 4 times in cell extract.
In many embodiment:, the part that one or more sequences that coding improves the protein that wood sugar or pectinose utilize can be used as the mosaic gene comprising this promotor is expressed.Coding region can be expressed in the xylose utilization approach comprising xylose isomerase, xylulokinase transketolase and transaldolase.These enzymes, their encoding sequence, and their participation xylose utilization approach are known.Particularly, as US7, any xylose isomerase disclosed in 998,722 (they are incorporated herein by reference) is the xylose isomerase by its encoding sequence be may be operably coupled to this promotor to express.
In addition, for the utilization of pectinose, effable coding region comprises L-arabinose isomerase (L-arabinose is changed into L-ribulose by it), L-ribulokinase (L-ribulose is become L-ribulose-5-phosphoric acid by it) and L-ribulose-5-phosphoric acid-4-epimerase (L-ribulose-5-phposphate is become D-xylulose by it), as US5,843, open in 760, it is incorporated herein by reference.In addition, the sequence that coding improves the other oroteins of xylose utilization can be expressed, such as ribose-5-phosphate isomerase (RPI), its catalysis ribulose-5-phosphoric acid and ribose-5-phposphate and belong to that to classify as EC5.3.1.6 (open in US20120156746, it is incorporated herein by reference) enzyme group, and ribulose-phosphoric acid-3-epimerase (RPE), its catalysis D-ribulose-5-phosphoric acid and D-xylulose-5-phposphate and belong to that to classify as EC5.1.3.1 (open in US20130157331, it is incorporated herein by reference) enzyme group.
In one embodiment, this promotor is for expressing the heterologous nucleotide acid molecule of encoding xylose isomerase, and it is for guiding the expression of the xylose isomerase improved compared with the expression from described ZmPgap.The Pgap of described improvement and xylose isomerase coding region form mosaic gene, and it usually also comprises 3 ' and stops control region.Termination control region can come from several genes and usually take from target host cell is natural gene.This area that is structured in of mosaic gene is known.
Any xylose isomerase coding region can be used in mosaic gene to express xylose isomerase with the PgapU by improving.Xylose isomerase belongs to EC5.3.1.5 group.US7,998,722 and US20110318801 in disclose the example of spendable suitable xylose isomerase zymoprotein and encoding sequence, it is incorporated in full by reference.
example
The present invention will set forth in the following examples further.Should be appreciated that, although these examples describe preferred embodiment of the present invention, only provide in an exemplary manner.By above-mentioned discussion and these examples, those skilled in the art can determine essential feature of the present invention, and not departing under the prerequisite in the spirit and scope of the invention, can carry out variations and modifications to adapt to multiple use and condition to the present invention.
basic skills
The implication of abbreviation is as follows: " kb " refers to kilobase, " bp " refers to base pair, " nt " refers to Nucleotide, " hr " refers to hour, " min " refers to minute, " sec " refers to second, " d " refers to number of days, " L " refers to liter, " ml " or " mL " refers to milliliter, " μ L " refers to microlitre, " μ g " refers to microgram, " ng " refers to nanogram, " mg " refers to milligram, " mM " refers to that milli rubs, " μM " refers to micro-rubbing, " nm " refers to nanometer, " μm ol " refers to micromole, " pmol " refers to picomole, " XI " refers to xylose isomerase, " nt " refers to Nucleotide.
Standard recombinant dna used herein and molecule clone technology are known in the art, and have in such as Publication about Document and more fully describe: Sambrook, J., Fritsch, and Maniatis E.F., T., MolecularCloning:ALaboratoryManual, 2nd edition, ColdSpringHarborLaboratory:ColdSpringHarbor, NY (1989) (hereinafter referred to as " Maniatis ") and Silhavy, T.J., Bennan, and Enquist M.L., L.W., ExperimentswithGeneFusions, ColdSpringHarborLaboratory:ColdSpringHarbor, NY (1984), and the people such as Ausubel, F.M., CurrentProtocolsinMolecularBiology, is published by GreenePublishingAssoc.andWiley-Interscience, Hoboken, NJ (1987)
example 1
promotor-GUS tests the structure of integrative vector
In order to be incorporated into by DNA in described zymomonas mobilis gene, build described pZP13 double cross (DCO) suicide vector.Fig. 1 illustrates its schematic diagram.But this plasmid has the skeleton of pBluescript, comprises intestinal bacteria replication site does not have zymomonas mobilis replication site.Therefore, it cannot breed in zymomonas mobilis.Synthesized by zymomonas mobilis genomic dna with PCR at pZP13 two the fragment DP1341-U and DP1341-D determining genome recombination that hit.Mentioned sequence is in described zymomonas mobilis gene order: the people such as Seo (2005) Nat.Biotechnol.23:63-68.NCBI reference number: NC_006526.2.1,951-bpDP1341-U fragment (SEQIDNO:8) comprises the front 217bp (from nt-1 to nt-217) of ZMO1250 encoding sequence and the upstream sequence of 1,734-bp.1,255-bpDP1341-D fragment (SEQIDNO:9) comprises the rear 704bp (from nt-218 to nt-921) of ZMO1250 encoding sequence and the downstream sequence of 551-bp.ZMO1250 encoding sequence in these two fragment mediates integrated transgene between them to described zymomonas mobilis genome between nt-217 and nt-218.ZMO1250 is positioned at from 1,355,129 to 1, the open reading frame of the zymomonas mobilis gene order of 356,049, the putative protein of its coding 306-aa.
PZP13 is also through constructing to comprise chimeric GUS expression cassette, and it is intersegmental that it is positioned at described target stator, and it is used as the reporter gene measuring the sub-intensity of test starting.It is by the test starting subsequence (Pro) defined with SpeI and NsiI site, 1,815-bp beta-glucuronidase encoding sequence (GUS; SEQIDNO:10) and 166-bp intestinal bacteria araD3 ' UTR (ECaraD3 ' UTR; SEQIDNO:11) form.Be positioned at 1, the 014-bpSpec-R expression cassette (SEQIDNO:12) in addition that described target stator is intersegmental, it selects the zymomonas mobilis cell transformed, side joint loxPw fragment for using Trobicin.The flank loxPw sequence comprising loxPw site and restriction site is SEQIDNO:15 and 16.
Described zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene (P for expressing test starting of described GUS coding region
gap) and the derivative of this promotor, P
gapU.P
gapstrong 305-bp glycolytic promoters (SEQIDNO:1), before this for restructuring Zymomonas mobilis strain in express xylose utilization genes.P
gapU305-bpP
gap(SEQIDNO:2) derivative, it has the change from C to T at position 90 place of described promoter sequence.The pZP13 suicide vector called after comprising these promotors driving GUS to express comprises P
gappZP1332 (SEQIDNO:13) and comprise P
gapUpZP1337 (SEQIDNO:14).
example 2
the integration of promotor-GUS expression cassette
Described test starting-GUS mosaic gene is incorporated in the genome of described wild-type Zymomonas mobilis strain ZW1 (ATCC#31821).Vibrate at MRM3G5 (1% yeast extract, 15mMKH with 150rpm at 30 DEG C
2pO
4, 4mMMgSO
4with 50g/L glucose) in overnight growth ZW1 prepare competent cell.ShimadzuUV-1200 spectrophotometer (Kyoto, Japan) is used to measure described OD
600value.Collecting cell is also resuspended in fresh culture to OD
600value is 0.05.Cell grows to logarithmic phase (OD early under similarity condition
600close to 0.5), then collect and wash 2 times with icy water and use 10% ice-cold glycerine washing once.The competent cell that collection obtains also is resuspended in 10% ice-cold glycerine to OD
600value is close to 100.Because the conversion of zymomonas mobilis needs non-methylated DNA, enter in intestinal bacteria SCS110 competent cell (Stratagene, LaJolla, CA) so first pZP1332 and pZP1337 is transformed.For each conversion, by a bacterium colony of institute's transformant in 10mLLB-Amp100 (the LB liquid nutrient medium containing 100mg/L penbritin) in 37 DEG C of grow overnight.QIAprepSpinDNAMiniprepKit (Qiagen, Valencia, CA) is used to prepare DNA from every 10mL culture.
The ZW1 competent cell of the non-methylated plasmid DNA of about l μ g and 50 μ L is mixed in 1MM electroporation cup (VWR, WestChester, PA).BT720TransporaterPlus (BTX-Genetronics, SanDiego, CA) is used to be entered in described cell by described plasmid DNA electroporation under 2.0KV.Make transformant at 30 DEG C at 1mLMMG5 substratum (50g/L glucose, 10g/L yeast extract, 5g/L Tryptones, 2.5g/L (NH
4)
2sO
4, 2g/LK
2hPO
4and 1mMMgSO
4) in recovery 4 hours, and with AnaeroPack (MitsubishiGasChemical at 30 DEG C, NewYork, NY) anaerobism wide-necked bottle in the upper growth of MMG5-Spec250 plate (there is the MMG5 of the Trobicin of 250mg/L and the agar of 15g/L) 3 days.Obtain the clone of described anti-Trobicin and streak inoculation on fresh MMG5-Spec250 plate, and grow under similarity condition as above, this shows that described chimeric GUS/Spec-R transgenic constructs has been incorporated in the genome of ZW1.In order to determine the incorperation and expression of described GUS expression cassette further, in the Falcon pipe that 14mL is with cover, transformant is inoculated into 5mLMRM3G5-Spec200 (there is the MRM3G5 of 200mg/L Trobicin) and in 30 DEG C, overnight growth under 150rpm oscillating condition.By 100 μ L cell culture fluid centrifugations in 1.5mL Eppendorf tube.1mL0.1MpH7.0 phosphate buffered saline buffer is used to wash the cell of described collection and be then resuspended in 100 μ LGUS staining fluids (1mL solution comprises 500 μ L0.2M sodium phosphate buffers, pH7.0,20 μ L0.5MEDTA, pH8.0,10 μ L10%tritonX-100,20 μ L50mg/mLX-Glue dimethyl formamide solutions, 450 μ L water).By carrying out color reaction in suspension described in night incubation at 37 DEG C.This reaction is stopped by removing described GUS solution and adding 100 μ L70% dehydrated alcohols.Measure according to significant blueness, a lot of transformant has the reaction of GUS stained positive.Therefore, these transformants not only have the integration of the construct of chimeric GUS/Spec-R, also express GUS and Spec-R transgenosis simultaneously.These transformant called afters ZW1-ZP1332 and ZW1-ZP1337.
example 3
the comparison of promotor is carried out by GUS activation analysis
In order to measure GUS activity, the two kinds of transformants comprising ZW1-ZP1332 (ZW1-ZP1332#11 and #12) or ZW1-ZP1337 (ZW1-ZP1337#1 and #8) are in 2mLMRM3G5-Spec200,30 DEG C, incubated overnight under 150rpm oscillating condition.Then 50 μ L nutrient solutions are added the fresh MRM3G5-Spec200 of 5mL and in 14mL Falcon pipe with cover in 30 DEG C, under 150rpm oscillating condition growth 16 hours until OD
600close to 6.By centrifugal collecting cell, analyze benchmark damping fluid (50mMNaPO with 1mLGUS
4damping fluid, pH7.0,1mMEDTA) wash twice, be resuspended to (GUS comprising 5% glycerine analyzes benchmark damping fluid) in 1mL protein extraction buffer.Each cell suspending liquid is placed in containing vibrating 3 times upper 20 second at BIO101FastprepFP1200 in the 5th step in the BIO1012mL of cracking matrix pearl (BIO101, LaJolla, CA) pipe with cover.Centrifugation cell fragment and pearl in microcentrifuge, and collect supernatant as protein extract.
Use PierceCoomassieProteinAssaySolution (ThermoFisherScientificInc., Rockford, IL) measures the protein concn in described extract.In described analysis, the protein extract of 10 μ L standard BSA solution (comprising 0.025,0.05,0.1 and 0.2 μ g/ μ L) and suitably dilution is mixed with the ProteinAssaySolution of 200 μ L in 96 orifice plates, and at room temperature hatches 5 minutes.VictorIII (PerkinElmer, Waltham, MA) measures OD
595and calculate protein concn by described equipment according to the typical curve of gained.Table 1 is the summary of protein concn in all extracting solutions.
protein concn in table 1 cell extract
The scheme for GUS activity analysis is adjusted: use described GUS as the reporter gene (SeanR.Gallagher edits, AcademicPress, Inc., 1992) of genetic expression according to GUS scheme.Improved for 96 orifice plates.In described analysis, described protein extraction buffer (GUS with 5% glycerine analyzes benchmark damping fluid) is used described ZW1-ZP1332#11 and #12 extracting solution of protein to be diluted 20 times and described ZW1-ZP1337#1 and #8 is diluted 80 times.By by 3,930 μ LGUS analyze benchmark damping fluids, and 20 μ L1MDTT and the mixing of 50 μ L100mMp-nitrophenyls-β-D-glucuronide (PNPG) are prepared fresh GUS analytical work damping fluid and be preheating to 37 DEG C.By by 200 μ L0.4MNa
2cO
3stop bath joins in each hole and prepares Falcon96 hole flat underside.In 0.2mLPCR pipe, carry out assembly GUS analytical reaction by the GUS working buffer liquid of the extracting solution of protein and 100 μ L preheatings that add 25 μ L dilutions, and hatch under 37 DEG C of conditions subsequently.At 15,30,45 and 60 minutes, from described reaction tubes, shift out 25 μ L reaction solutions and mix with 200 μ L stop baths in ready 96 orifice plates immediately.Three groups of parallel reactors are carried out to each extracting solution of protein.Xiang Kongzhong adds the mixture of 20 μ LGUS analytical work damping fluids and 5 μ L protein extraction buffer as blank.After reacting completely, in VictorIII plate reading machine, scan described 96 orifice plates to measure the OD in each hole
405.The reading of described blank is deducted from each reaction.By the average OD of the 4-nitrophenol that reflection discharges through GRD beta-glucuronidase
405map relative to the reaction times, produce instruction per minute OD
405slope S (the OD of change
405/ minute).Finally, speed of response R (OD is calculated based on described reaction slope S and the protein content joined in reaction
405/ ug protein/minute).Reaction slope (S) and the speed of response (R) of each protein extract is provided in following table 2.Use average response speed to calculate specific activity, its instruction is relative to P
gappromotor intensity.Result proves described promotor P
gapUpromotor P described in the strength ratio had
gapheight about 4.4 times.
table 2.P
ganU
and P
gap
relative Promoter Strength
Claims (13)
1. the nucleic acid molecule of a synthesis, the nucleic acid molecule of described synthesis comprises the zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter having and replace the base of C at the position 90 place T of natural motion fermentation single cell bacterium (Z.mobilis) glyceraldehyde-3-phosphate dehydrogenase gene promoter, and the sequence of described natural motion fermentation single cell bacterium glyceraldehyde-3-phosphate dehydrogenase gene promoter is shown in SEQID1.
2. a nucleic acid molecule for synthesis, the nucleic acid molecule of described synthesis comprise be selected from SEQIDNO:2,4, the zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter sequence of 6 and 7.
3. a mosaic gene, described mosaic gene comprises the nucleic acid molecule of the synthesis described in the claim 1 or 2 that may be operably coupled to exogenous nucleic acid molecule.
4. mosaic gene according to claim 3, wherein said exogenous nucleic acid molecule coded protein or peptide.
5. mosaic gene according to claim 3, wherein said exogenous nucleic acid molecule coding is selected from the rna regulation molecule of sense-rna, ribozyme and RNA interfering.
6. a carrier, described carrier comprises the nucleic acid molecule of the synthesis described in claim 1 or 2.
7. the method for a transforming bacterial cells, described bacterial cell is selected from fermentation single cell bacterium (Zymomonas) cell and fermentation bacillus (Zymobacter) cell, and described method comprises and being incorporated in described cell by the nucleic acid molecule of the synthesis described in claim 1 or 2.
8. method according to claim 7, wherein introduces and comprises in the nucleic acid molecule integrates of the synthesis described in claim 1 or 2 to the genome of described cell or remain on plasmid that described cell inner stablity copies.
9. a recombinant bacteria bacterial strain, described recombinant bacteria bacterial strain comprises the nucleic acid of the synthesis described in claim 1 or 2, and wherein said bacterial isolates is selected from fermentation single cell bacterium and fermentation bacillus.
10. recombinant bacteria bacterial strain according to claim 9, the nucleic acid molecule of wherein said separation is the promotor of mosaic gene.
11. recombinant bacteria bacterial strains according to claim 10, wherein said mosaic gene comprises the nucleic acid molecule of separation, and the nucleic acid molecule encoding of described separation is selected from following enzyme: xylose isomerase, xylulokinase, transketolase, transaldolase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphoric acid-4-epimerase, ribose-5-phosphate isomerase and ribulose-phosphoric acid-3-epimerase.
12. 1 kinds of methods for the production of the synthetic glycerine aldehyde-3-phosphate dehydrogenase gene promoter improved, described method comprises:
A) be always separated with the glyceraldehyde-3-phosphate dehydrogenase gene of fermentation bacillus the nucleic acid fragment comprising glyceraldehyde-3-phosphate dehydrogenase gene promoter region from fermentation single cell bacterium; And
B) by position 90 place T, the base of C is replaced in the fragment of the separation be incorporated into a), thus produce the synthetic glycerine aldehyde-3-phosphate dehydrogenase gene promoter improved.
Synthetic glycerine aldehyde-3-the phosphate dehydrogenase gene promoter of 13. 1 kinds of improvement prepared by method according to claim 12.
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| US14/037666 | 2013-09-26 | ||
| PCT/US2014/057386 WO2015048243A1 (en) | 2013-09-26 | 2014-09-25 | High expression zymomonas promoters |
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| EP (1) | EP3049513A1 (en) |
| JP (1) | JP2016531555A (en) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852631A (en) * | 2019-02-14 | 2019-06-07 | 湖北大学 | A kind of varying strength ribosome bind site and promoter screening technique and element |
| CN109913487A (en) * | 2019-02-14 | 2019-06-21 | 湖北大学 | A method for identification of biological elements based on dual fluorescent reporter gene system |
| WO2020164195A1 (en) * | 2019-02-14 | 2020-08-20 | 湖北大学 | Method for identifying biological elements based on dual fluorescent reporter gene system and biological element library constructed based on same |
| CN118652307A (en) * | 2024-06-05 | 2024-09-17 | 武汉睿嘉康生物科技有限公司 | Proteins for display on the surface of Zymomonas mobilis |
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| JP6152127B2 (en) * | 2015-02-16 | 2017-06-21 | 日東電工株式会社 | Polarizer, polarizing plate and image display device |
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| CN101981184A (en) * | 2008-03-27 | 2011-02-23 | 纳幕尔杜邦公司 | High expression zymomonas promoters |
| CN102985540A (en) * | 2010-06-29 | 2013-03-20 | 纳幕尔杜邦公司 | Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity |
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| US5514583A (en) | 1994-04-15 | 1996-05-07 | Midwest Research Institute | Recombinant zymomonas for pentose fermentation |
| US5843760A (en) | 1994-04-15 | 1998-12-01 | Midwest Research Institute | Single zymomonas mobilis strain for xylose and arabinose fermentation |
| US5712133A (en) | 1994-04-15 | 1998-01-27 | Midwest Research Institute | Pentose fermentation by recombinant zymomonas |
| US7223575B2 (en) | 2000-05-01 | 2007-05-29 | Midwest Research Institute | Zymomonas pentose-sugar fermenting strains and uses thereof |
| US6566107B1 (en) | 2000-11-01 | 2003-05-20 | Midwest Research Institute | Recombinant Zymomonas mobilis with improved xylose utilization |
| AU2009228323B2 (en) | 2008-03-27 | 2014-01-30 | Alliance For Sustainable Energy, Llc | Zymomonas with improved xylose utilization |
| US8623623B2 (en) | 2010-06-29 | 2014-01-07 | E I Du Pont De Nemours And Company | Xylose utilization in recombinant Zymomonas |
| US8911983B2 (en) | 2011-12-20 | 2014-12-16 | E I Du Pont De Nemours And Company | Pnp gene modification for improved xylose utilization in Zymomonas |
-
2013
- 2013-09-26 US US14/037,666 patent/US9139818B2/en not_active Expired - Fee Related
-
2014
- 2014-09-25 BR BR112016006560A patent/BR112016006560A2/en not_active IP Right Cessation
- 2014-09-25 WO PCT/US2014/057386 patent/WO2015048243A1/en active Application Filing
- 2014-09-25 JP JP2016517413A patent/JP2016531555A/en not_active Abandoned
- 2014-09-25 CN CN201480051856.XA patent/CN105555947A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101861385A (en) * | 2006-09-28 | 2010-10-13 | 纳幕尔杜邦公司 | Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production |
| CN101981184A (en) * | 2008-03-27 | 2011-02-23 | 纳幕尔杜邦公司 | High expression zymomonas promoters |
| CN102985540A (en) * | 2010-06-29 | 2013-03-20 | 纳幕尔杜邦公司 | Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852631A (en) * | 2019-02-14 | 2019-06-07 | 湖北大学 | A kind of varying strength ribosome bind site and promoter screening technique and element |
| CN109913487A (en) * | 2019-02-14 | 2019-06-21 | 湖北大学 | A method for identification of biological elements based on dual fluorescent reporter gene system |
| WO2020164195A1 (en) * | 2019-02-14 | 2020-08-20 | 湖北大学 | Method for identifying biological elements based on dual fluorescent reporter gene system and biological element library constructed based on same |
| US11248258B2 (en) | 2019-02-14 | 2022-02-15 | Hubei University | Method for characterizing biological part based on dual-fluorescent reporter gene system and biological part library constructed thereon |
| CN118652307A (en) * | 2024-06-05 | 2024-09-17 | 武汉睿嘉康生物科技有限公司 | Proteins for display on the surface of Zymomonas mobilis |
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| EP3049513A1 (en) | 2016-08-03 |
| JP2016531555A (en) | 2016-10-13 |
| WO2015048243A1 (en) | 2015-04-02 |
| US9139818B2 (en) | 2015-09-22 |
| AU2014326594A1 (en) | 2016-03-10 |
| US20150087070A1 (en) | 2015-03-26 |
| BR112016006560A2 (en) | 2017-09-12 |
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